Method for measuring the vitality of cells

ABSTRACT

A method is described for non-destructive measurement of vitality of biological cells, especially for determination of apoptosis, in which the at least one cell is exposed to high-frequency alternating, especially rotating, electric fields and/or impedance test fields, and at least one rotation measurement, one dielectrophoresis measurement and/or one impedance measurement is performed with the cell for at least one frequency range or individual frequencies, from which at least one measurement parameter is determined that is characteristic of the vitality state of the cell.

BACKGROUND OF THE INVENTION

The invention relates to a method for the non-destructive measurement ofthe vitality of biological cells, especially for non-destructivedetermination of apoptosis of cells. The method according to theinvention furthermore allows the distinction of apoptotic cells fromvital cells and necrotically damaged cells. Furthermore, the presentinvention relates to a method for identification of substances whichinfluence apoptosis.

In nature, cell death occurs in two different manifestations, asnecrosis and apoptosis. Necrosis is cell death caused by an unspecificphysical or chemical damage. By injury of the barrier function of theplasma membrane, its semipermeability for ions (especially Ca²⁺, Na⁺,K⁺) is disturbed. This results in massive ion and solution influx intothe cell, which causes the cell to swell and finally rupture. Thus,compartmentation of the cell is abolished and the cell content isspilled. Subsequently, this process causes inflammatory reactions in theorganism. Usually, necrotic damages take place within seconds tominutes.

In contrast to this, apoptosis is a programmed cell death. Apoptosis isan organized process, which is needed during the development andmaintenance of an organism to eliminate unwanted or damaged cellswithout harming the organism in total. It is controlled by receptors(glucocorticoid receptor, TNFR, Fas, NGFR) and usually depends on denovo protein synthesis. Cellular proteases (ICE, caspases) andendonucleases become activated. Since almost all cells of an organismcan undergo apoptosis, the trigger of the apoptotic process must beunder permanent and strict physiological control. From the purephenomenological viewpoint, apoptosis can be divided into six successivestages: 1. shrinkage, 2. zeiosis (plasma membrane protuberances), 3.chromatin collapse, 4. collapse of the cellular nucleus, 5.fragmentation of the nucleus into smaller units and fragmentation ofcellular DNA, 6. separation of apoptotic vesicles (Thompson, C. B.,1995, Apoptosis in the pathogenesis and treatment of disease, Science267: 1456-1462). Throughout the entire process, the barrier function ofthe plasma membrane is maintained. The triggering of apoptosis occursthrough both internal signals of the organism as well as throughexternal influences, such as radiation, chemical substances or reagents.Apoptosis is a process the first signs of which may develop only hourslater. In contrast to necrotic cells, apoptotic cells are recognized andremoved by neighbouring cells or macrophages (Orrenius, S., 1995,Apoptosis: molecular mechanisms and implications for human disease, J.Internal Medicine 237: 529-536). Here, no inflammatory reaction occurs.

Newer studies demonstrate that a multitude of diseases is based ondisturbances in the regulation of apoptosis. Diseases associated withblockage of apoptosis include forms of cancer, such as carcinomas withp53 mutations and hormone-dependent tumours; autoimmune diseases, suchas rheumatoid arthritis, diabetes mellitus; and viral infections, suchas infections with herpes viruses, pox viruses and adenoviruses.Diseases which may be caused by an increased rate of apoptosis includeparticularly AIDS; neurodegenerative disorders, such as Alzheimer'sdisease, Parkinson's disease; the effects of toxins, such as liverdiseases from large amounts of alcohol (Thompson, C. B., 1995, Apoptosisin the pathogenesis and treatment of disease, Science 267: 1456-1462).

For this reason, a multitude of methods based on apoptosis-specificparameters have been developed for determination of apoptosis. Thesemethods, for example, are based on the determination of thephosphatidylserine portion of the extracellular cell surface by annexinV binding, determination of hypoploid nuclei, determination of cytosoliccytochrome C or caspase concentration, or the determination of caspaseactivation.

Detection is performed by microscopic, fluorescence-based andbiochemical procedures. Electron microscopy and phase contrastmicroscopy, for example, detect the morphological appearance duringapoptosis, such as shrinkage, membrane protuberances, and the presenceof micronuclei (S. Verhaegen 1998, Microscopical Study of Cell Death viaApoptosis, European Microscopy and Analysis). However, quantification ofapoptosis in a sample by microscopy requires a high degree of judgementon the part of the investigator, and manual counting of the cells afteroptical evaluation, or suitable image processing software.

A further well-known method for detection of apoptosis in a sample ofsuspended cells requires the use of a flow cytometer. Here, fluorescencedyes staining DNA are used (K. H. Elstein and R. M. Zucker 1994,Comparison of Cellular and Nuclear Flow Cytometric Techniques forDiscriminating Apoptotic Subpopulations, Experimental Cell Research 211,322-331), or a biochemical method is employed in which the process ofDNA fragmentation is visualized by incorporation of nucleotides withfluorescent labels (e.g. TUNNEL method, R. S. Douglas, A. D. Tarshis, C.H. Pletcher, P. C. Nowell and J. S. Moore, 1995, A simplified method forthe coordinate examination of apoptosis and surface phenotype of murinelymphocytes, Journal of Immunological Methods 188, 219-228). Also,specific fluorescently labelled probes are used for cell surfacemolecules. An example of such a probe is fluorescenctly labelled annexinV (G. Koopman, C. P. M. Reutlingsperger, G. A. M. Kuijten, R. M. J.Keeshen, S. T. Pals and M. H. J. van Oers, 1994, Annexin V for FlowCytometric Detection of Phosphatidylserine Expression on B CellsUndergoing Apoptosis, Blood 84,5, 1415-1420) which binds tophosphatidylserine on the cell surface and thus visualizes therestructuring of the plasma membrane in the early apoptotic stage.However, all flow-cytometric methods for the measurement of apoptosisrequire calibration of the method and the system. In addition, a highnumber of cells (approx. 10⁶) is required for analysis.

Other methods for the detection of apoptosis require the destruction ofall cells in a sample and the gel-electrophoretic or biochemicaldetection of DNA fragmentation (M. Leist, F. Gantner, I. Bohlinger, P.G. Germann, G. Tiegs and A. Wendel, 1994, Murine Hepatocyte ApoptosisInduced In Vitro and In Vivo by TNF-a Requires Transcriptional Arrest,The Journal of Immunology 153, 1778-1788). These biochemical methods areprocedures with several steps in which several reagents are used. Thisresults in a very long time of analysis of up to 6 hours. In addition,several hundred cells are needed for one analysis.

Due to the relevance of disturbed regulation of apoptosis in associationwith a multitude of diseases, the study of apoptosis is also a centralpart of the search for new pharmaceutical agents, of the evaluation oftheir activity against diseases as well as in environmental analytics.Generally, a compound which influences apoptosis must not only becapable of modulating apoptosis but also of permeating the intact cellmembrane. Furthermore it is assumed that a multitude of cellularreceptors, proteins, cell components and cofactors influences theprocess of apoptosis in living cells.

Therefore, a multitude of screening methods has already been developedfor the identification of substances which influence apoptosis, whichare based on the determination of apoptosis-specific parameters in thecell or in cell-free systems.

For example, WO 98/02579 describes a screening assay for theidentification of apoptosis-regulating substances in a cell-free system.Here, several apoptosis-specific parameters, such as cytochrome c orCPP32 protease activity, are determined as measures of apoptosis. Forscreening, a supernatant obtained at 100,000×g from the cytosol ofnon-apoptotic cells is employed. The determination of substancesinfluencing apoptosis was performed by addition of test compounds tothis supernatant which may have negative, positive or no effect onapoptosis. The results obtained in this way are compared to those of areference substance.

WO 98/55615 describes a screening assay for determination oftherapeutically active substances influencing apoptosis. Cell-freeassays are described which are designed to study compounds that competewith cytochrome c for binding to apaf-1. Furthermore, assays fordetermination of substances influencing apoptosis are also describedwhich are based on the study of the proteolytic cleavage of caspase-3precursors in presence or absence of potential substances influencingapoptosis.

In WO 99/18856, a method is described for the detection of substancesfor induction or inhibition of enzymes of the apoptosis cascade,especially caspases. The method is performed with whole cells ortissues. In order to do so, a portion of the cells is treated with thetest compounds and fluorescenctly labelled reporter molecules, whereasanother fraction is treated only with the reporters, as control. Achange in fluorescence compared to the control sample indicates that asubstance to be tested influences enzymes of the cascade.

Taken together, all currently known methods for determination ofapoptosis or apoptosis-modulating substances have a multitude ofdisadvantages. Thus, they frequently require a great deal of time due toelaborate experimental steps and in addition require large quantitiesof, for example, antibodies, dyes and other reagents that can enter thecell. Furthermore, most of the methods are based on final cell lysis,eventually leading to destruction of the cells. Other investigatedparameters, such as e.g. annexin V, are not specific for apoptosis,still other methods require a large number of cells. Furthermore, manymethods are only suited to study late stages of apoptosis whichfrequently do not occur in vivo, since the apoptotic cells have alreadybeen taken up by neighbouring cells or macrophages by phagocytosis inthis stage.

Frequently used cell-free assays, such as the determination ofcytochrome c and other apoptosis-specific markers, are not suitable fordetermination of the ability of substances affecting apoptosis to passthrough the intact cell membrane.

Furthermore it is though that different cell types have differentreceptors and cofactors for modulation of apoptosis. For this reason itis not possible to find specific or organ-specific apoptosis modulatorswith cell-free assays.

Due to the fact that in most of the known methods the importance ofcellular receptors and other cofactors is neglected, the danger ofidentification of false-positive or false-negative substancesinfluencing apoptosis is high, since these substances do not show any ordo not show the expected effect in living cells. In addition, substancesinfluencing apoptosis that modulate apoptosis indirectly through one ofthese receptors or cofactors are not detected.

Considering these disadvantages of the known methods, it would bedesirable to have a method available that is suitable for theunambiguous identification of apoptosis and determination of substanceswhich modulate apoptosis. This method should be able to recognizeapoptosis in an early stage, and should be easily performable, even withwhole cells.

Dielectrophoretic techniques, such as e.g. the dielectric singleparticle spectroscopy (R. Pethig and G. H. Markx, 1997, Applications ofdielectrophoresis in biotechnology, Trends in Biotechnology 15, 426-432)and electrorotation (T. Schnelle, T. Müller and G. Fuhr, 1999,Dielectric single particle spectroscopy for measurement of dispersion,Medical & Biological Engineering & Computing 37, 264-271) are alreadyknown as such.

Also, devices for the measurement of electrorotation in combination withthe use of optical tweezers are already known (T. Schnelle, T. Müller,C. Reichle and G. Fuhr, 2000, Combined dielectrophoretic field cages andlaser tweezers for electrorotation, Applied Physics B, Lasers andOptics, Springer-Verlag) and automatic signal uptake (C. Reichle, T.Müller, T. Schnelle and G. Fuhr, 1999, Electro-rotation in octopolemicro cages, Journal of Physics D: Applied Physics 32, 2128-2135; DeGasparis, Wang, Yang, Becker and Gascoyne, 1998, Meas. Sci. Technol. 9,518-529).

The object of the present invention is to provide an improved method fornon-destructive measurement of vitality, especially for the detection ofapoptosis, in biological cells. The invention is intended to allow theprovision of an improved method for identification of substancesinfluencing apoptosis.

SUMMARY OF THE INVENTION

The basic idea of the invention is the provision of a method fornon-destructive measurement of vitality of cells by introducing at leastone cell to be examined in a microsystem, in which the at least one cellis exposed to high frequency electric rotating fields or electricalternating fields, especially impedance test fields.

This method particularly allows for differentiation between vital cells,necrotic cells and cells that are in the state of apoptosis.

The invention includes in its preferred embodiments methods fordetection of apoptosis in suspended cells by dielectrophoretictechniques.

DETAILED DESCRIPTION OF THE INVENTION

A distinction especially between vital, necrotic and apoptotic cells isnot possible with the usual described methods.

Furthermore, the method according to the invention has many advantagescompared to the methods that are already known. For example, thenecessity of specific detection reagents as well as the resulting timeconsumed in preparing the samples by processes that frequently includeseveral steps, and elaborate image analysis are eliminated. In addition,the method according to the invention is characterized by singlecell-sensitivity, specificity for the apoptotic process, and allows fordistinction between necrotic and apoptotic cells, the detection and theautomatic and fast determination of apoptosis at an early stage. For thedetermination, the cells solely are to be present in a suspended state.The addition of one or more reagents is not required. The effort insample preparation is therefore very small. The method is not limitedwith respect to the number of cells; the procedure can be performed withindividual cells. The individual measurement is very fast (e.g. 30seconds for electrorotation) and recording a time course in a singlecell sample or a single cell is possible. In addition, the method has nomeasurable influence on the vitality of the cells.

The method according to the invention for non-destructive determinationof apoptosis at an early stage is characterized by the at least one cellbeing introduced to a microsystem in which the at least one cell isexposed to high frequency alternating fields, especially rotatingelectric fields and/or impedance test fields.

It is preferred that at least one rotational measurement,dielectrophoretic measurement and/or impedance measurement is performedwith the cell for at least one frequency range or individualfrequencies, resulting in determination of at least one measurementparameter which is characteristic for the vitality state of the cell.

In another embodiment of the method according to the invention, at leastone rotational measurement, dielectrophoretic measurement and/orimpedance measurement is performed with the minimum of a single cell forat least two frequency ranges or at least two frequencies, resulting inat least two values for the measurement parameter, this minimum of twovalues being compared to each other, thus leading to a determination ofthe state of vitality of the cell, particularly if it is a cell in thestate of apoptosis, necrosis or if it is a vital cell. Hereby it isespecially preferred that the measurement parameter comprises rotationspeed, migration speed, electrophoretic mobility and/or impedance,especially amplitude and/or phase.

The embodiment described here has the particular advantage that only aminimum of two values have to be determined for one measurementparameter, and therefore a significant amount of time is saved comparedto the recording of an entire spectrum. Therefore, this embodiment isespecially suited for high throughput screening.

Especially preferred is the determination of a rotation spectrum of thecell by measuring the rotation speed (inverse rotation time) of the atleast one cell based on the frequency of the rotating electric field,whereby it is desirable that the at least one cell is concomitantly heldin the focus of an optical trap, especially optical tweezers, duringrecording of the rotation speed. The rotation measurement is performedpreferably with cells suspended in physiological solutions. Theconductivity of the solution is preferably in the range of 1 . . . 1.6S/m.

However it is not necessary to record complete spectra. In fact it issufficient to perform the measurements with one or more fixedfrequencies of the rotating field, preferably with one frequency each ofthe frequency range of 1 to 4 MHz, preferably 2 to 3 MHz, especiallypreferred 2.3 to 2.6 MHz and the frequency range 5 to 100 MHz,preferably 6 to 50 MHz, especially preferred 8 to 15 MHz.

To determine if an examined cell is in the state of apoptosis, therotation speed determined in the higher frequency range is put inrelation to the rotation speed determined in the lower frequency range.If the ratio obtained is above 1, especially preferred 1.1 to 1.8, thecell to be analyzed is an apoptotic cell. For necrotic cells, the ratiois below 1, especially preferred 0.6 to 0.8, and for vital cells it isequal to 1.

For determination of apoptosis it is also possible to compare therotation spectrum of the at least one cell to be analyzed with therotation spectrum of a reference cell, the reference cell possibly beinga vital cell, preferably of the same cell type as the cell to beanalyzed. However, it is also desirable that the reference cell is acell which has specifically been transferred to the state of apoptosisand which is preferably of the same cell type as the cell to beanalyzed, or that the reference cell is a cell which has specificallybeen transferred to the state of necrosis and which is preferably of thesame cell type as the cell to be analyzed.

The state of apoptosis can then be preferably determined by the rotationspectrum of the cell to be analyzed in contrast to the vital or necroticreference cell or comparable with the apoptotic reference cell havingits maximum in the frequency range of 5 to 100 MHz, preferably 6 to 50MHz, especially preferred 8 to 15 MHz.

Another embodiment of the method according to the invention is based onthe different behaviour of normal, necrotic and apoptotic cells indielectrophoresis (DEP).

For determination of apoptosis according to the invention, it maytherefore be also desirable to detect a dielectrophoresis spectrum,preferably in the frequency range of 0 to 5 MHz, especially preferred of1 to 4 MHz, by measuring the migration speed or the electrophoreticmobility of the at least one cell in dependence on the frequency of theelectric field, whereby in a special embodiment the at least one cellbeing held in focus of an optical trap. According to the invention,apoptosis is detected by the frequency change of the transition fromnegative to positive dielectrophoresis in the dielectrophoresis spectrumbeing in the range of 3.3 to 3.8 MHz, preferably at 3.5 MHz.

However it may also be desirable to determine the migration speed or theelectrophoretic mobility of the at least one cell only with at least 2frequencies of the frequency range of 0-5 MHz, preferably 1-4 MHz.

If the frequency crossing of the transition from negative to positivedielectrophoresis is 2.8 to 3.1 MHz, preferably 3 MHz, the cell to beanalyzed is a vital cell; if it is 1.8 to 2.2 MHz, preferably 2 MHz, thecell to be analyzed is a necrotic cell.

In addition, apoptosis may be determined by the dielectrophoresisspectrum and/or the at least 2 migration speeds or electrophoreticmobilities of the at least one cell to be analyzed being compared to thedielectrophoresis spectrum and/or the at least 2 migration speeds orelectrophoretic mobilities of a reference cell, the reference cellpossibly being a vital cell, preferably of the same cell type as thecell to be analyzed. However, it is also desirable that the referencecell is a cell which has been specifically transferred to the state ofapoptosis, preferably a cell of the same cell type as the cell to beanalyzed, or that the reference cell is a cell which has beenspecifically transferred to the state of necrosis, preferably a cell ofthe same cell type as the cell to be analyzed.

In a further embodiment of the present invention, the different forceswhich act upon vital, apoptotic and necrotic cells in an alternatingelectric field are used to separate the different cell types. Theincrease in the internal conductivity and the dielectric constant ofnecrotic cells compared to vital cells results in modified values of thereal part of the electric dipole moment. The force acting on necroticcells compared to vital and apoptotic cells is markedly decreased in PBSbuffer preferably in the frequency range over 1 MHz (FIG. 5), and thus,dielectric separation is possible.

A decrease in the electrolyte content of the buffer solution below arange around 0.3 S/m results preferably in a frequency range up to 3 MHzfor apoptotic and necrotic cells in a lower negative DEP compared tovital cells. In contrast to this, a strongly decreased positive DEP ofapoptotic and normal cells compared to necrotic cells develops in afrequency range of 3 to 120 MHz. The frequency crossing at thetransition from negative to positive DEP is here in the range from 3.3to 3.8 MHz for apoptotic cells, preferably 3.5 MHz, for vital cells inthe range of 2.8 to 3.1 MHz, preferably 3 MHz, and for necrotic cells inthe range of 1.8 to 2.2 MHz, preferably 2 MHz. Thus, an effectiveseparation of all three cell types is possible by dielectrophoreticforces. In this way, the proportions of necrotic, apoptotic and vitalcells of a sample can be determined (FIG. 5B).

According to another embodiment of the method according to theinvention, the determination of apoptosis may also be performed bymeasurement of the impedance property, especially amplitude and/orphase, of the at least one cell based on the frequency of the impedancetest field. An impedance spectrum is determined, the at least one cellpreferably being held in focus by an optical trap. It is also desirablethat the impedance of individual cells is determined betweenmicroelectrodes in small volumes. Furthermore it is preferred that theimpedance of the at least one cell is recorded at at least twofrequencies in the frequency range of 1 Hz to 100 kHz, preferably 0.5kHz to 10 kHz. The impedance of a cell suspension is calculated asfollows:

${Z(\omega)} = \frac{1}{\left( {\frac{1}{R_{ext}} + \frac{1}{R_{c} + \frac{1}{1\;\omega\; C_{M}}}} \right)}$(Fricke, H., 1923, The electric capacity of cell suspensions, Phys. Rev.21, 708-709), with R_(ext) being the external solution resistance, R_(c)being the cytoplasm resistance and C_(M) being the membrane capacity.For an accurate analysis of biological cells, complex models arerequired which reflect the capacity and Ohmic portions of allcompartments (Irimajiri, A., Suzaki, T., Asami, K. and Hanai, T., 1991,Dielectric Modeling of Biological Cells. Models and Algorithm, Bull.Inst. Chem. Res. Kyoto Univ. 69/4, p. 421-438).

In order to guarantee high sensitivity of the measurement according tothe invention, it is desirable that the contribution of the solution1/R_(ext) is small compared to the contribution of the individual cell.This applies either to small frequencies or for a small distance betweenthe cell and the measuring electrodes which are also small (single cellimpedance measurement), the single impedance measurement fordetermination of differences in cytoplasmic properties in the megahertzrange being especially desirable. In addition, the cellularmodifications during apoptosis result in a modified impedance signal inthe lower frequency range: 1 Hz-100 kHz, preferably 0.5 kHz-10 kHz. Fordetermination of apoptosis it is therefore preferred to record impedancespectra in this frequency range.

For determination of apoptosis it is also possible that the impedancespectrum and/or the at least 2 impedances (value and/or phase) of the atleast one cell to be analyzed is compared with the impedance spectrumand/or the at least 2 impedances (value and/or phase) of a referencecell, the reference cell possibly being a vital cell of preferably thesame cell type as the cell to be analyzed. However, it is also desirablethat the reference cell is a cell which has been specificallytransferred to the state of apoptosis, preferably a cell of the samecell type as the cell to be analyzed, or that the reference cell is acell which has been specifically transferred to the state of necrosis,preferably a cell of the same cell type as the cell to be analyzed.

The state of apoptosis can be then preferably be determined by theimpedance spectrum and/or the at least 2 impedances of the cell to beanalyzed in contrast to the vital or necrotic reference cell, orcomparable to the apoptotic reference cell show changes especially inthe frequency range of 1 Hz-100 kHz, preferably 0.5 kHz-10 kHz.

To follow the change over time of the properties of the at least onecell, especially for determination of the time course of apoptosis, itmay be desirable that the rotation spectrum and/or the at least 2rotation speeds and/or the dielectrophoresis spectra and/or the at least2 migration speeds or electrophoretic mobilities and/or the impedancespectra and/or the at least 2 impedances are recorded in dependence ontime.

In a preferred embodiment of the method according to the invention, thefollowing procedure is provided:

-   -   Providing at least one cell sample to be analyzed,    -   Introduction of the at least one cell of the cell sample to be        analyzed in a microsystem and detection of the at least one        measurement parameter which is characteristic for the vitality        condition of the cell,    -   Separation of the cells of the cell sample to be analyzed or a        certain volume of this cell sample according to their vitality        state.

In summary, the method according to the invention is especially suitedfor diagnosis and/or therapeutic control of diseases and/or processeswhich are associated with an increase in the apoptosis rate, such asparticularly AIDS, neurodegenerative diseases, particularly Alzheimer'sdisease, Parkinson's disease; liver diseases caused by toxins,particularly alcohol, diseases resulting from a hormone production orsecretion deficiency.

According to the invention it is also possible to use the method fordetection of apoptosis for diagnosis and/or therapeutic control ofdiseases and/or processes which are associated with a decrease in theapoptosis rate, such as particularly malignant and benignhyperproliferative diseases such as cancer, hormone-dependent tumours,leukaemia, autoimmune diseases, particularly arthritis, diabetesmellitus and viral infections, particularly those caused by herpesviruses, pox viruses or adenoviruses.

The method according to the invention furthermore allows a rapidperformance, and low number of cells is needed for analysis. The methodcan even be performed with a single cell. It is therefore very wellsuited for therapy control particularly of malign and benignhyperproliferative diseases during chemotherapy, radiation therapy,immunotherapy, surgery or a combination of these therapies.

According to the invention it was also possible to provide a method thatallows for the identification of substances which influence apoptosis,taking into account the cellular receptors and other cofactors, andwhich therefore reduces the risk of identification of false-positive orfalse-negative substances influencing apoptosis.

A method according to the invention for identification ofapoptosis-influencing substances is characterized particularly by thefollowing steps:

-   -   Providing of at least one cell culture sample to be analyzed,    -   Addition of a potential substance influencing apoptosis or        mixtures of at least two of these substances,    -   Introduction of at least one cell of the cell culture sample to        be analyzed in a microsystem and determination of at least one        measurement parameter which is characteristic for the vitality        state of the cell, the at least one cell being exposed to high        frequency, alternating, particularly rotating, electrical fields        and/or impedance test fields.    -   Determination of the effects on apoptosis, especially by        comparing the behaviour of this at least one cell in the        electrical field with the behaviour of the at least one cell of        a reference sample.

In a preferred embodiment, at least two values are determined for onemeasurement parameter, and the effects on apoptosis are determined bycomparing these at at least two values. Furthermore, the effects onapoptosis are determined by comparing at least one value for onemeasurement parameter of the at least one cell with the at least onevalue of the at least one cell of a reference sample.

It is preferred to use, as the reference sample, a sample of the samecell type as the sample to be analyzed before addition of the at leastone potential substance influencing apoptosis.

In another embodiment of the method according to the invention, thebehaviour of the at least one cell of the cell culture sample to beanalyzed and the at least one cell of the reference sample are examinedin the electrical field by recording a rotation spectrum by measurementof the rotation speed of each cell in dependence on the frequency of therotating electrical fields.

The effects on apoptosis of at least one substance can be detectedaccording to the invention by the rotation spectrum of the at least onecell to be analyzed of the sample in contrast to the spectrum of the atleast one cell of the reference sample demonstrating a change especiallyin the frequency range of 5 to 100, preferably 6 to 50 MHz, especiallypreferred 8 to 15 MHz and/or in the frequency range of 1 to 4 MHz,preferably 2 to 3 MHz, especially preferably 2.3 to 2.6 MHz.

It may, however, also be preferred that the rotation speed of the cellsof the cell sample and the reference is determined only at at least onefrequency each of the frequency range of 1 to 4 MHz, preferably 2 to 3MHz, especially preferred 2.3 to 2.5 MHz, and the frequency range 5 to100 MHz, preferably 6 to 50 MHz, especially preferred 8 to 15 MHz, therotation speed determined at the higher frequency range being put inrelation to the rotation speed which is determined in the lowerfrequency range.

For an apoptotic cell, the ratio is greater than 1, especially preferred1.1 to 1.8; for a necrotic cell smaller than 1, especially preferred 0.6to 0.8 and for a vital cell equal to 1.

However, it may also be desirable to examine the behaviour of the atleast one cell of the cell culture sample to be analyzed and the atleast one cell of the reference sample in the electric fields byrecording a dielectrophoresis spectrum by measurement of the migrationspeed or the dielectric mobility of each cell based in the frequency ofthe alternating electrical fields.

a further embodiment of the method, the effects on apoptosis of the atleast one substance are determined by the frequency change from negativeto positive dielectrophoresis in the dielectrophoresis spectrum of theat least one cell to be analyzed on contrast to the spectrum of the atleast one cell of the reference sample being in the range of 3.3 to 3.8MHz, preferably 3.5 MHz, the dielectrophoresis spectrum of the at leastone cell to be analyzed being recorded in the frequency range of 0 to 5MHz, preferably 1 to 4 MHz.

In a further embodiment of the method according to the invention, thebehaviour of the at least one cell of the cell culture sample to beanalyzed and the at least one cell of the reference sample in theelectric field is examined by recording an impedance spectrum bymeasurement of the impedance properties (value and/or phase) of eachcell based on the frequency of the impedance test field.

A further embodiment of the method describes that the effects onapoptosis of the at least one substance can be determined by changesoccurring in the frequency range of 1 Hz to 100 kHz, preferably 0.5 kHzto 10 kHz in the impedance spectrum of the at least one cell to beanalyzed in contrast to the spectrum of the at least one cell of thereference sample.

During recording of the rotation spectrum at at least one frequencyand/or a dielectrophoresis spectrum and/or an impedance spectrum, it maybe desirable that the at least one cell is held in the focus of anoptical trap.

For determination of the influence of potential substances influencingapoptosis it may be preferred to determine the proportion of apoptoticcells in a sample. For this purpose, a separation of the cells of acertain sample volume and/or of a certain volume of the reference samplemay be conducted first. The separation can be conducteddielectrophoretically. Then, the number of apoptotic cells of the sampleto be analyzed is determined in relation to the number of apoptoticcells in the reference sample.

Thus, substances which increase apoptosis are characterized by anincrease in the number of apoptotic cells in relation to the referencesample.

Substances that do not influence apoptosis are characterized by a numberof apoptotic cells that is comparable to the reference sample.

Thus, substances which inhibit apoptosis are characterized by a smallernumber of apoptotic cells in relation to the reference sample.

Since the natural apoptosis rate in cell cultures often is very small,it may also be desirable in determining potential apoptosis-inhibitingsubstances to add an apoptosis-increasing substance to the cell culturesample to be analyzed, either before or during the addition of the atleast one substance to be analyzed, the cell culture sample which hadbeen supplemented with an apoptosis-increasing substance and/or a cellcultures sample containing the same amount of apoptosis-increasingsubstance being used as reference sample.

A substance which decreases apoptosis is characterized by containing asmaller number of apoptotic cells in relation to the reference sample.

In summary, the determination of apoptosis according to the presentinvention allows for the determination of apoptosis with a simple,efficient, fast and high throughput method by recognizing early stagesof apoptosis without destroying the cell. A further advantage of themethod according to the invention is that this method can be applied tocells in their physiological environment without changing the propertiesof the cells or its surface. The methods for determination of substancesinfluencing apoptosis are capable of exactly measuring their ability toenter the cell, taking into account the influence of receptors andcofactors. The risk of a false-positive or false-negative determinationof substances influencing apoptosis is significantly lower compared tothe known methods. In addition, it is possible to determine cell type-or organ-specific modulators. Furthermore, no specific cellmembrane-permeating fluorescent dyes are required for the application ofthe method according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows light-microscopic images of a normal (A) and an apoptotic(B) Jurkat cell (clone E6.1 from the European Collection of Animal CellCultures, Salisbury, England). The apoptotic cell was treated with 5μg/ml actinomycin D for 4 hrs.

FIG. 2 shows electrorotation spectra of a normal Jurkat cell (A), aJurkat cell that has been treated with 10 μm ionomycin for approx. 5 min(B), a Jurkat cell that has been damaged dielectrophoretically with 50kHz and Erms=30 kV/m for 20 seconds (C), an RMA cell that has been heldwith 30 mW of optical tweezers of 1080 nm wavelength for 40 min (thymomacell line from the clone C57BL/6 from Professor Peter Walden, Charité,Berlin) (D) and a Jurkat cell that has been treated for 4 hours withactinomycin D (E).

In FIG. 3, the acceleration of the electrorotation and uptake ofpropidium iodine during necrotic damaging of Jurkat cells by increase ofvoltage in a dielectrophoretic octupole field cage is shown. (A)rotation speed (inverse rotation time) of a Jurkat cell at 4.3 V, (B)Frequency of rotation of the same Jurkat cell after increasing thevoltage to 6.8 V. (C) Propidium iodine fluorescence of the Jurkat cellat 4.3 V, (D) Propidium iodine fluorescence of the same Jurkat cellafter increasing the voltage to 6.8 V. Each time, the electrorotationwas measured with 4.3 V. Three different cells were analyzed.

FIG. 4 shows the propidium iodine fluorescence of a normal U937 cell(A), an apoptotic U937 cell (monocytic cell line from the EuropeanCollection of Animal Cell Cultures, Salisbury, England) treated withactinomycin D for 4 hours (B), and a necrotic U937 cell (C).

In FIG. 5, electrorotation and dielectrophoresis of T lymphocytes(Jurkat) based in the field frequency are shown. Curves a, b and cbelong to normal, necrotic and apoptotic cells, respectively. In A,automatic measurements of the electrorotation in PBS (conductivity 1.5S/m) as well as curve fits according to multishell spherical models withdispersion are shown. B shows the corresponding dielectrophoreticbehaviour, with the curves' indices corresponding to different externalconductivities (1: 0.05 S/m, 2: 0.3 S/m, 3: 1.5 S/m (PBS)).

The dispersive material behaviour is described as the sum of relaxations(dielectric constant ε, conductivity σ, angular frequency of theelectrical field ω, relaxation time of the dispersion τ, number ofdispersions η):

$\begin{matrix}{ɛ = {ɛ_{\infty} + {\frac{1}{ɛ_{vac}}{\sum\limits_{k = 1}^{n}\frac{\tau_{k}\Delta\;\sigma_{2}}{1 + \left( {\omega\;\tau_{k}} \right)^{2}}}}}} & {with} & {{\Delta\; ɛ} = {{- \Delta}\;\sigma}}\end{matrix}$

FIG. 6 shows a lateral view of a microsystem for electrorotationmeasurements in microchannels (C. C. Reichle, T. Schnelle, T. Müller, T.Leya and G. Fuhr, 2000, A new microsystem for automatic electrorotationmeasurements using laser tweezers, Biochimica et Biophysica Acta 1459,p. 218- 229).

EXAMPLE 1 Electrorotation Measurement

Materials Used

-   Jurkat cells: Clone E6.1 from the European Collection of Animal Cell    Cultures, Salisbury, England;-   U937 cells: Monocytic cell line from the European Collection of    Animal Cell Cultures, Salisbury, England;-   RMA cells: Thymoma cell line from the clone C57/BL6 from Professor    Peter Walden, Charité, Berlin, Ref: C. Reichle, K. Sparbier, T.    Müller, T. Schnelle, P. Walden and G. Fuhr, in press, Combined laser    tweezers and dielectric field cage for the analysis of multivalent    receptor ligand interactions on single cells, ELECTROPHORESIS,    Miniaturization II;-   Actinomycin D: SIGMA Aldrich GmbH, Steinheim, Germany;-   Ionomycin: Calbiochem, Bad Soden, Germany;-   Propidium iodine: SIGMA Aldrich GmbH, Steinheim, Germany;-   PBS: Phosphate buffered saline, with calcium and magnesium,    Seromed/Biochrom, Berlin, Germany;-   RPMI 1640 medium: HEPES buffered, GIBCO Life Technologies,    Karlsruhe, Germany;-   Fetal calf serum: Seromed/Biochrom, Berlin, Germany;-   Penicillin: Seromed/Biochrom, Berlin, Germany;-   Inositol: SIGMA Aldrich GmbH, Steinheim, Germany.    Instrument:

An instrument already described by C. C. Reichle, T. Schnelle, T.Müller, T. Leya and G. Fuhr (2000, A new microsystem for automaticelectrorotation measurements using laser tweezers, Biochimica etBiophysica Acta 1459, p. 218- 229), a combination of dielectrophoreticalfield cages with optical traps, especially laser tweezers, was used fordetermination of the electrorotation spectra (FIG. 6). A comparabledevice is also described in T. Schnelle, T. Müller, C. Reichle and G.Fuhr 2000, Combined dielectrophoretic field cages and laser tweezers forelectrorotation, Applied Physics B, Lasers and Optics, Springer Verlag.

Procedure:

Jurkat T lymphoma cells and U937 monocytic cells were cultivated inRPMI-1640 medium supplemented with 10% fetal calf serum and 100 IU/mlpenicillin as well as 100 IU/ml streptomycin. Apoptosis was triggered byaddition of 5 μg/ml actinomycin D in a culture that was seeded the daybefore with 2×10⁵ cells per ml. The classical morphological changesduring apoptosis were documented photographically (see FIG. 1). Atdifferent times, an aliquot was taken from the culture, added to amixture of isotonic phosphate buffer (PBS, D8662 from SIGMA with calciumand magnesium) and, if desired, 0.3 M inositol with correspondinglyindicated electrical conductivity, and introduced in a device formeasurement of the electrorotation in a microelectrode system. As acontrol, untreated Jurkat cells were used (FIG. 5). For comparison,electrorotation spectra (ER spectra) were determined fromionomycin-treated cells (10 μg/ml ionomycin) as well as from cells theplasma membrane of which had been damaged by increasing the voltage from4 V to 6.8 V or a treatment with optical tweezers with 30-60 mW (2.8 Aof the 1 W Nd:YAG laser, TEM₀₀, 1064 nm, LD 3000i, Laser Quantum Ltd.,Manchester, UK, P.A.L.M. System) for 15 minutes. For this, the cellswere transferred individually to the center of the quadrupole oroctupole of the microelectrode system. For this, optical tweezers with apower of approx. 30 mW were used in the indicated cases at the site ofholding. The frequency of the cell rotation was measured eitherautomatically or using a timer (FIG. 2). The uptake of propidium iodine(5 μm) in necrotically damaged cells or comparative measurements withvital and apoptotic cells (FIGS. 3 and 4) was measured by confocalnanofluorometry (Gradl, T. Müller, C. Reichle, T. Schnelle and G. Fuhr,1999, Micro-Electrode Systems for Cell Analysis, European Journal ofCell Biology, Suppl. 49, Vol. 78; T. Schnelle, T. Müller, G. Gradl, S.G. Shirley and G. Fuhr, 2000, Dielectrophoretic manipulation ofsuspended submicron particles, ELECTROPHORESIS 2000, 21, p. 66-73).

Electrorotation Measurement:

The device for measurement of the electrorotation was attached to afluorescence correlation spectrometer. The cells were made visible overa 40×W 1.2 NA lens. They were excited with 1.5 μW by a helium-neon laserwith a wavelength of 543 nm and the radiated fluorescence was passedthrough a 585 DF 35 band pass filter over a 50 μm pinhole to aphotodiode, with which the fluorescence photons were counted.

Result:

The characteristic ER spectrum of a normal (A) Jurkat cell shown in FIG.1 demonstrates a wide peak of 1.5 to 7 MHz, during which a maximalrotation speed of the cell is measured, with a maximum around 2.5 MHz(FIG. 2A). A cell treated with the ionophore ionomycin (Calbiochem, BadSoden, Germany) gives a markedly different ER spectrum from a normalcell (FIG. 2B). Ionomycin at a higher concentration induces the uptakeof calcium from the extracellular space into the cell (J. B. Smith, T.Zheng and R. M. Lyu, 1989, Ionomycin releases calcium from thesarcoplasmic reticulum and activates Na⁺/Ca²⁺ exchange in vascularsmooth muscle cells, Cell Calcium 10, p. 125-134). At the ionomycinconcentration of 10 μM, the fits of the corresponding spectra show thatin addition, uptake of water and probably also of buffer ions insubstantial amounts, such as sodium and potassium, takes place. This canbe seen clearly from the increase in conductivity σ (as sum of σ₀, Δσ₁and Δσ₂) from 0.77 to 1.15 s m⁻¹ and the dielectric constant ε_(ω) from45 to 75 in the cell (C. Reichle, T. Schnelle, T. Müller, T. LEya and G.Fuhr, 2000, A new microsystem for automated electrorotation measurementsusing laser tweezers, Biochimica et Biophysica Acta 1459, p. 218-229).They approximate the values of the surrounding buffer PBS.

Since the dielectrically active total concentration of sodium andpotassium ions of a cell before the treatment is lower than the ionconcentration of the PBS (phosphate buffered saline, with calcium andmagnesium, Seromed/Biochrom, Berlin, Germany), approx. 0.54 S/m vs. 1.5S/m; a net influx of ions and water occurs and the cell swells. In theER spectrum, this is reflected in a great enlargement of the 2.5 MHzpeak, and can be explained by an approx. 40% increase in the HFconductivity and a more than 60% increase of the dielectric constant ofthe cytoplasm. Cells that have experienced necrotic damage byapplication of an increased voltage in the dielectric field cage showvery similar behavior (FIG. 2C). This dielectric breakthrough isassociated with damage of the plasma membrane, which can be made visibleby the uptake of the DNA dye propidium iodine (FIG. 3). Subsequentlyions can exchange between the internal cellular space and thesurrounding buffer here as well. The resulting ER spectrum is verysimilar to the spectrum of a cell treated with ionomycin. A necroticdamage with optic tweezers also results in the same phenomenon (FIG. 2D). All described damage up to here is associated with a loss of theintegrity of the plasma membrane and an approach of the ion content ofthe internal to that of the external cellular spaces. If theconductivity of the external solution decreases to a value below theconductivity of the cell, a net efflux of ions would decrease theelectrolyte content of the cell. This results in a further decrease inthe frequency with the fastest rotation (f_(c)) and enhances thedifferences compared to the untreated cells. Surprisingly, acharacteristic spectrum of an apoptotic cell is different from thespectrum of a normal cell or the spectrum of a necrotically damaged cell(FIG. 2E). The peak at 2.5 MHz disappears completely. The highestrotation speed occurs at approx. 10 MHz. A typical apoptotic cell withthis described ER spectrum has strong plasma protuberances (see FIG.1B). In this stage, however, its plasma membrane is still intact (FIG.4). Thus, the method according to the invention is suited to distinguishbetween normal, apoptotic and necrotically damaged cells.

Electrorotation measurements at the two frequencies 2.5 MHz (FIG. 2F)and 10 MHz (FIG. 2G) can already demonstrate clearly the differencebetween these three different cell states. The ratio between therotation speed (inverse rotation time) of the measurement at 10 MHz andthe measurement at 2.5 MHz is for the normal cell near 1 (with adeviation of approx. 10%). For the necrotically damaged cell it isclearly smaller than 1 (0.6-0.8) and for the apoptotic cell it is higherthan 1 (in this case 1.4).

1. A method for non-destructive measurement of three different vitality states of biological cells, comprising the steps of: exposing the cells to high-frequency, alternating electric fields or impedance test fields, determining a first rotation speed of at least one cell at at least one first frequency within a first frequency range from 1 to 4 MHz, determining a second rotation speed of the at least one cell at at least one second frequency within a second frequency range from 5 to 100 MHz, and determining a quotient of the second rotation speed divided by the first rotation speed, wherein said quotient is characteristic for at least one of the three different vitality states of the cell, so as to identify at least one cell in a state of apoptosis, at least one cell in a state of necrosis, and at least one cell in a vital state.
 2. The method according to claim 1, wherein an apoptotic cell is determined by said quotient being greater than one, a necrotic cell is determined by said quotient being less than 1, and a vital cell is determined by said quotient being equal to
 1. 3. The method according to claim 2, wherein the quotient for apoptotic cells is 1.1 to 1.8, and the quotient for necrotic cells is 0.6 to 0.8.
 4. The method according to claim 1, wherein said apoptosis is determined by at least two rotation speeds of at least one cell to be analyzed being compared to at least two reference rotation speeds of a reference cell determined at the same frequencies as the cell to be analyzed.
 5. The method according to claim 4, wherein the reference cell is of the same cell type as the cell to be analyzed, and is a vital cell, a cell that has been specifically placed in an apoptotic state, or a cell that has been specifically placed in a necrotic state.
 6. A method of identifying apoptosis-influencing substances, said method comprising the following steps: providing at least one cell culture sample to be analyzed, addition to the at least one cell culture sample of at least one substance potentially influencing apoptosis, introduction of the at least one cell culture sample to be analyzed into a microsystem for determining rotation speeds of the at least one cell, exposing the at least one cell of the cell to high-frequency, alternating electric fields or impedance test fields, determining a first rotation speed of at least one cell at at least one first frequency within a first frequency range from 1 to 4 MHz, determining a second rotation speed of the at least one cell at at least one second frequency within a second frequency range from 5 to 100 MHz, and determining a quotient of the second rotation speed divided by the first rotation speed, wherein said quotient is characteristic for at least one of the three different vitality states of the cell, so as to identify whether the at least one cell is in a state of apoptosis, a state of necrosis, or a vital state, and to thereby identify whether the at least one substance influences apoptosis.
 7. The method according to claim 6, wherein prior to addition of the at least one potential apoptosis-influencing substance, a sample of a same cell type as the sample to be analyzed is used as a reference sample.
 8. The method according to claim 6, wherein an apoptotic cell is determined by said quotient being greater than 1, a necrotic cell is determined by said quotient being less than 1, and a vital cell is determined by said quotient being equal to
 1. 9. The method according to claim 8, wherein the quotient for apoptotic cells is 1.1 to 1.8, and the quotient for necrotic cells is 0.6 to 0.8. 